ATM Influences the Efficiency of TCRβ Rearrangement,Subsequent TCRβ-Dependent T Cell Development,and Generation of the Pre-Selection TCRβ CDR3 Repertoire

Generation and resolution of DNA double-strand breaks is required to assemble antigen-specific receptors from the genes encoding V, D, and J gene segments during recombination. The present report investigates the requirement for ataxia telangiectasia-mutated (ATM) kinase, a component of DNA double-strand break repair, during TCRβ recombination and in subsequent TCRβ-dependent repertoire generation and thymocyte development. CD4⁻CD8⁻ double negative stage 2/3 thymocytes from ATM-deficient mice have both an increased frequency of cells with DNA break foci at TCRβ loci and reduced Vβ-DJβ rearrangement. Sequencing of TCRβ complementarity-determining region 3 demonstrates that ATM-deficient CD4⁺CD8⁺ double positive thymocytes and peripheral T cells have altered processing of coding ends for both in-frame and out-of-frame TCRβ rearrangements, providing the unique demonstration that ATM deficiency alters the expressed TCRβ repertoire by a selection-independent mechanism. ATMKO thymi exhibit a partial developmental block in DN cells as they negotiate the β-selection checkpoint to become double negative stage 4 and CD4⁺CD8⁺ thymocytes, resulting in reduced numbers of CD4⁺CD8⁺ cells. Importantly, expression of a rearranged TCRβ transgene substantially reverses this defect in CD4⁺CD8⁺ cells, directly linking a requirement for ATM during endogenous TCRβ rearrangement to subsequent TCRβ-dependent stages of development. These results demonstrate that ATM plays an important role in TCRβ rearrangement, generation of the TCRβ CDR3 repertoire, and efficient TCRβ-dependent T cell development.     

A TCRβ Repertoire Signature Can Predict Experimental Cerebral Malaria

Cerebral Malaria (CM) is associated with a pathogenic T cell response. Mice infected by P. berghei ANKA clone 1.49 (PbA) developing CM (CM⁺) present an altered PBL TCR repertoire, partly due to recurrently expanded T cell clones, as compared to non-infected and CM^- infected mice. To analyse the relationship between repertoire alteration and CM, we performed a kinetic analysis of the TRBV repertoire during the course of the infection until CM-related death in PbA-infected mice. The repertoires of PBL, splenocytes and brain lymphocytes were compared between infected and non-infected mice using a high-throughput CDR3 spectratyping method. We observed a modification of the whole TCR repertoire in the spleen and blood of infected mice, from the fifth and the sixth day post-infection, respectively, while only three TRBV were significantly perturbed in the brain of infected mice. Using multivariate analysis and statistical modelling, we identified a unique TCRβ signature discriminating CM⁺ from CTR mice, enriched during the course of the infection in the spleen and the blood and predicting CM onset. These results highlight a dynamic modification and compartmentalization of the TCR diversity during the course of PbA infection, and provide a novel method to identify disease-associated TCRβ signature as diagnostic and prognostic biomarkers.     

Statistical Methods for Integrating Randomized Clinical Studies for Evaluation of Combination Treatment Therapy

In regulatory applications, evaluation of a combination drug at a fixed dose is based on pairwise comparisons of the combination with its component drugs. These comparisons generate the least expected gain of the combination relative to its components as the key effect parameter for evaluation. Two test methods are developed for the evaluation to combine multiple clinical studies that are deemed combinable. One test method is based on a linear combination of the Min tests from individual studies. In the other test method, weighted estimators are first derived for the pairwise comparisons between the combination and the respective components by combining the studies. A Min test is then applied to these estimators. The latter test method tends to be more powerful than the former test method. A test-based confidence interval is constructed for the least expected gain of the combination relative to its components. A test for heterogeneity across studies is also developed.     

Characterization of human αβTCR repertoire and discovery of D-D fusion in TCRβ chains

The characterization of the human T-cell receptor (TCR) repertoire has made remarkable progress, with most of the work focusing on the TCRβ chains. Here, we analyzed the diversity and complexity of both the TCRα and TCRβ repertoires of three healthy donors. We found that the diversity of the TCRα repertoire is higher than that of the TCRβ repertoire, whereas the usages of the V and J genes tended to be preferential with similar TRAV and TRAJ patterns in all three donors. The V-J pairings, like the V and J gene usages, were slightly preferential. We also found that the TRDV1 gene rearranges with the majority of TRAJ genes, suggesting that TRDV1 is a shared TRAV/DV gene (TRAV42/DV1). Moreover, we uncovered the presence of tandem TRBD (TRB D gene) usage in ～2% of the productive human TCRβ CDR3 sequences.     

Comparative Evaluation of Three Supplements for Helicobacter pylori Growth in Liquid Culture

Helicobacter pylori, a microaerophilic fastidious bacterium, has been cultured on various plating and broth media since its discovery. Although the agar media can be sufficient for the identification, typing, and antibiotic resistance studies, no secretory antigen of H. pylori can be evaluated in such media. Thus, satisfactory growth of H. pylori in liquid culture which is needed for analysis of secretory proteins without the presence of interfering agents is in demand. We assessed the impact of β-cyclodextrin, Fetal Bovine Serum (FBS), and charcoal as supplements for H. pylori growth. Furthermore, we aimed to identify the most favorable supplement that supports the secretion of the dominant secretory protein, vacuolating cytotoxin (VacA). Five clinical strains were cultured on broth media and the growth, viability, morphology, and protein content of each strain were determined. Our results revealed that β-cyclodextrin supports the growth rate, viability, and cell lysate protein content to the extent similar to FBS. Application of β-cyclodextrin is found to postpone spiral to coccoid conversion up to 72 h of incubation. Although FBS supports a higher VacA protein content, presence of interfering macromolecules in FBS questions its utility particularly for purposes of studying extra cellular proteins such as VacA. This study recommends further application of β-cyclodextrin as a culture supplement with the potential capacity in neutralizing toxic compounds and flourishing the secretion of H. pylori proteins without addition of interfering proteins.     

Non–Culture-Based Methods for the Diagnosis of Invasive Candidiasis

Given the limitations of current fungal diagnostics, the use of non–culture-based methods for the diagnosis of invasive candidiasis (IC) is highly warranted. The implementation of molecular diagnostic strategies could permit the timely onset of appropriate therapy and may be expected to pave the way for improved clinical outcome of IC. Polymerase chain reaction (PCR) may have higher sensitivity for the diagnosis of IC than conventional blood cultures. The detection of fungal antigens generally requires a large fungal burden, and the presence of fungus-specific antibodies may not correlate with the underlying diseases. Therefore, the combined mannan and anti-mannan antibody testing is recommended. No single test has been shown convincingly to compensate for all the limitations of culture. Real-time PCR coupled with fungal culture and/or antigen detection will likely be required to significantly ameliorate the diagnostic problems in IC.     

Evaluation of the Redesigned Enterotube—a System for the Identification of Enterobacteriaceae

The redesigned Enterotube has been evaluated with 414 unknown Enterobacteriaceae cultures from the stock culture collection of the Center for Disease Control. When the Enterotube was used as recommended by the manufacturer, an average of 96.4% of these cultures were correctly identified. Only two groups (Salmonella and Edwardsiella) were identified with less than 90% accuracy (89.2 and 87.5%, respectively). The Enterotube now provides a convenient, rapid, and accurate test system for the identification of typically reacting enteric bacteria.     

An Evaluation of β-Propiolactone for the Sterilization of Fermentation Media

Twenty-five bacterial species were cultured in basal broth plus 1 of 19 different carbohydrates which were sterilized by Seitz filtration, autoclaving (112 C, 10 min), or exposure to 0.2% β-propiolactone (BPL). No significant differences were found either in the visual observations for acid and gas, pH, or titrable acidity determinations after 3 days of incubation with any of the three preparations tested. An effort was made to further determine the effect of BPL and heat on carbohydrates by assaying for glucose before and after treatment. Results indicated that glucose was not degraded by 0.2% BPL, however, it was shown that autoclave temperatures caused extensive degradation. Statistical treatment of the results from Warburg studies indicated that BPL-treated glucose showed no appreciable toxic effects, although the actual oxygen uptake was not as great as with Seitz- or autoclave-treated glucose. The application of the BPL sterilization process was discussed.     

Evaluation of the immunogenicity of a single chain chimeric peptide composed of hCGβ and oLHα for inhibition of the growth of hCGβ-expressing cancer cells

Human chorionic gonadotropin (hCG) is a membrane-associated protein highly expressed in several types of human cancer cells. The expression in the cancer cells indicates that hCG may be a potential target molecule for cancer immunotherapy. The objective of this study was to develop a novel immunogenic molecule, which can efficiently induce the neutralizing antibody against hCG and which is also suitable for mass production. The immunogenicity of the recombinant single chain chimeric protein of hCGβ-oLHα expressed by yeast was examined. Additionally, the inhibitory effects of the anti-hCGβ-oLHα antibody on the growth of hCG-positive cancer cells were determined. It was found that hCGβ-oLHα yielded high titers of anti-hCG rabbit antibody that could effectively neutralize the bioactivity of hCG. The rabbit anti-hCGβ-oLHα IgG inhibited the proliferation of hCG-expressing human colorectal cancer cells (LS-174, HCT-116, HCT-15 and KM-12) in a dose-dependent manner. Furthermore, an intact anti-tumor vaccine was prepared by conjugating hCGβ-oLHα with tetanus toxoid (TT) and this was used to immunize Balb/c mice bearing hCG-expressing SP2/0 tumor cells. The progression of tumors in these immunized mice was remarkably inhibited. These results suggest that hCGβ-oLHα is a new promising immunogenic molecule for the development of an anti-hCG-based cancer vaccine.     

Membrane-mimetic systems for biophysical studies of the amyloid-β peptide

The interplay between the amyloid-β (Aβ) peptide and cellular membranes have been proposed as an important mechanism for toxicity in Alzheimer's disease (AD). Membrane environments appear to influence Aβ aggregation and may stabilize intermediate Aβ oligomeric states that are considered to be neurotoxic. One important role for molecular biophysics within the field of Aβ studies is to characterize the structure and dynamics of the Aβ peptide in various states, as well as the kinetics of transfer between these states. Because biological cell membranes are very complex, simplified membrane models are needed to facilitate studies of Aβ and other amyloid proteins in lipid environments. In this review, we examine different membrane-mimetic systems available for molecular studies of Aβ. An introduction to each system is given, and examples of important findings are presented for each system. The benefits and drawbacks of each system are discussed from methodical and biological perspectives.     

Comparative Evaluation of Five Culture Media with Triplex PCR Assay for Detection of Methicillin-Resistant Staphylococcus aureus

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) is responsible for nosocomial and community-acquired infections. Hence, rapid and accurate laboratory diagnosis of MRSA is a vital constituent of control measures. The present study evaluated five different methods for the identification of MRSA. A total of 207 S. aureus clinical isolates that consisted of 89 MRSA and 118 methicillin-susceptible S. aureus (MSSA) strains confirmed by PCR were tested. MRSA strains were evaluated by five different methods: chromogenic MRSA agar (CMRSA), oxacillin resistance screening agar base (ORSAB), mannitol salt oxacillin agar (MSO), mannitol salt cefoxitin agar with two different concentrations of cefoxitin [4 μg/ml (MSC-4) and 6 μg/ml (MSC-6)]. The results of the different methods were compared to mecA PCR as the gold standard. MSC-6 showed only six false-positive MRSA in comparison with PCR. The sensitivities and specificities of MSC-6, MSC-4, MSO-4, ORSAB, and CMRSA were as follows: 98.9/94.9%, 100/83.1%, 89.9/87.3%, 97.8/96.6%, and 95.5/94.9%, respectively. In comparison with PCR, it was found that both MSC-6 and ORSAB were relatively the least expensive screening tests (0.70 and0.70 and 1.00, respectively). In conclusion, all methods were comparable, but MSC-6 was the least expensive medium for MRSA screening.     

Computational Investigation of Pkcβ Inhibitors for the Treatment of Diabetic Retinopathy

Diabetic Retinopathy (DR) is one of the attenuating complications of diabetes mellitus. The key gene responsible for causing diabetic retinopathy is protein kinase C beta (PKCβ). Protein kinase C is a family of protein kinase enzymes which are involved in controlling the function of other proteins through phosphorylation mechanism and plays a crucial role in signal transduction mechanisms. Among all the PKC isoenzymes, PKCβ could be a significant isoenzyme involved in vascular dysfunction during hyperglycemia. Studies show that oral administration of PKCβ inhibitor Ruboxistaurin ({"type":"entrez-nucleotide","attrs":{"text":"LY333531","term_id":"1257370768","term_text":"LY333531"}}LY333531), decreases vessel permeability and improves retinal condition. Thus compounds that decrease the PKCβ activation would be helpful in the treatment of diabetic retinopathy. The compounds similar to Ruboxistaurin are taken from Super Target database and docking analysis was performed. Maleimide derivative 3 showed highest binding affinities compared to Ruboxistaurin and so we advise that compound may be utilized in the treatment of diabetic retinopathy.     

Binding studies of truncated variants of the Aβ peptide to the V-domain of the RAGE receptor reveal Aβ residues responsible for binding

Alzheimer's disease (AD) symptoms correlate with the concentration of soluble, although not necessarily monomeric forms of Aβ peptide in the brain parenchyma. The RAGE receptor has been implicated as the protein responsible for active transport of Aβ from blood circulation to the brain. In murine models of AD, inhibition of the Aβ:RAGE interaction decreases the levels of Aβ in the brain. Inhibition of the Aβ:RAGE interaction would be a promising alternative for the therapy of AD. Rational design of an Aβ:RAGE interaction blocker requires detailed knowledge of the structure of the complex. However, the binding domain of RAGE is natively unfolded in physiological conditions, which severely hampers the application of classic methods of protein structure analysis to the design of an antagonist. Here, alternative methods are used to characterize the structural properties of the RAGE-ligand binding domain and to monitor the binding of a series of truncated variants of Aβ. Using intrinsic RAGE tryptophan fluorescence and mass spectrometry of non-covalent protein-ligand complexes we have identified shorter versions of Aβ that bind to the RAGE V-domain. We have also shown in cell culture experiments that a selected shortened version of Aβ effectively inhibits full-length Aβ, RAGE-mediated, cell uptake. Thus, a truncated version of Aβ capable of blocking its receptor-mediated internalization was established, revealing the binding code and providing the lead compound in the process of drug design.     

High Algebraic Order Methods for the Numerical Solution of the Schrödinger Equation

Abstract

A family of new hybrid four-step tenth algebraic order methods with phase-lag of order 16(2)22 is developed for the numerical solution of the Schrödinger equation. Based on the new methods a variable-step procedure is introduced. Numerical illustrations obtained for the approximation of the phase shift problem for the well known case of the Lenard-Jones potential and for the numerical solution of the coupled equations arising from the Schrödinger equation show that these new methods are better than other finite difference methods.     

Comparison of the TCR ζ-chain with the FcR γ-chain in chimeric TCR constructs for T cell activation and apoptosis

A promising strategy for cancer treatment is adoptive gene therapy/immunotherapy by genetically modifying T cells with a chimeric T cell receptor (cTCR). When transduced T cells (T-bodies) specifically bind to tumor antigens through cTCR, they will become cytotoxic T lymphocytes (CTL) and lyse the tumor cells in a non-major histocompatibility complex (MHC)-restricted manner. Both the FcR gamma-chain and the TCR zeta-chain have been used to construct such cTCR, and both have shown specific cytolytic functions against tumor cells. However, most researchers believe that the zeta-chain generates stronger cytolytic activities against tumor than the gamma-chain and therefore would be a better candidate for cTCR construction. On the other hand, because of the lack of costimulation signaling in such constructs, the T-body might cause activation-induced T cell death (AICD) when bound to tumor antigens. Therefore, one can argue that the gamma-chain might generate less AICD than the zeta-chain because the gamma-chain has only one immunoreceptor tyrosine-based activation motif (ITAM), and the cytolytic activities can be therefore recycled. Two cTCR, GAHgamma and GAHzeta, were constructed and evaluated for cytokine production, specific cytolytic function and AICD in T-bodies after exposure to tumor cells. Using EGP-2-positive LS174T colorectal carcinoma cells as targets, there was no substantial difference observed between a gamma-chain or zeta-chain as the T-body signaling moiety in terms of specific cytolytic functions and induced cytokine production. This paper also demonstrates that, in the absence of a costimulation system, tumor antigen may not trigger apoptosis of T cells transduced with a cTCR carrying either an FcR gamma-chain or a TCR zeta-chain. These observations challenge current ideas about the role of ITAM in T cell activation.